GLF-1, a 37 amino acid peptide naturally formed by proteolysis of the 160 amino acid precursor protein preproglucagon, was first identified in 1987 as an incretin hormone. GLP-1 is secreted by the L-cells of the intestine in response to food ingestion and has been found to stimulate insulin secretion (insulinotropic action) causing glucose uptake by cells which decreases serum glucose levels (see, e g., Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)). GLP-1 is poorly active. A subsequent endogenous cleavage between the 6th and 7th position produces a more potent biologically active GLP-1(7-37)OH peptide. Approximately 80% of the GLP-1(7-37)OH so produced is amidated at the C-terminal in conjunction with removal of he terminal glycine residues in the L-cells and is commonly referred to GLP-1(7-36)NH2. Molecules which are reasonably homologous to, or are derived from, or based on these native forms will generally be referred to as GLP's in this specification.
The biological effects and metabolic turnover of the free acid, the amide form, and many of the numerous known GLP's are similar and show promise as agents for the treatment of diabetes, obesity, and related conditions, including but not limited to impaired glucose tolerance and insulin resistance. However, many GLP's suffer from extremely short biological half lives, some as short as 3-5 minutes, which makes them unattractive for use as pharmaceutical agents. Presently, the activity of dipeptidyl-peptidase-IV (DPP-IV) is believed to readily inactivate many GLP's and is in part responsible for the very short serum half lives observed. Rapid absorption and clearance following parenteral administration are also factors. Thus, there is a need to find a means for prolonging the action of these promising agents.
One such approach has been to modify these molecules to protect them from in vivo cleavage by DPP-IV. For example, see U.S. Pat. No. 5,512,549. In the insulin arts, it has long been known that extended time action can be achieved by administering crystalline protein formulations into the subcutis which act like depots, paying out soluble protein over time.
Heterogeneous micro crystalline clusters of GLP-1(7-37)OH have been grown from saline solutions and examined after crystal soaking treatment with zinc and/or m-cresol (Kim and Haren, Pharma. Res. Vol. 12 No. 11 (1995)). Also, crude crystalline suspensions of GLP(7-36)NH2 containing needle-like crystals and amorphous precipitation have been prepared from phosphate solutions containing zinc or protamine (Pridal, et. al., International Journal of Pharmaceutics Vol. 136, pp. 53-59 (1996)). Also, EP 0 619 322 A 2 describes the preparation of micro-crystalline forms of GLP-1(7-37)OH by mixing solutions of the protein in pH 7-8.5 buffer with certain combinations of salts and low molecular weight polyethylene glycols (PEG). However, such crystalline clusters and crude suspensions are less than ideal for preparing long acting pharmaceutical formulations of GLP's since they are loosely bound heterogeneous clusters of crystals or amorphous-crystalline suspensions which tend to trap impurities and are otherwise difficult to reproducibly manufacture and administer.
Most unexpectedly it was discovered that single tetragonal flat rod shaped or plate-like crystals Of various GLP's could be reproducibly formed from a mother liquor containing a GLP dissolved in a buffered solution and a C1-3 alcohol, or optionally a mono or disaccharide, over a wide range of pH conditions. The resulting single flat rod shaped or plate-like crystals are superior to, and offer significant advantages over, the GLP-1(7-37)OH crystal clusters or crude suspensions known in the art.
The single tetragonal flat rod shaped or plate-like crystals of the present invention are less prone to trap impurities and therefore may be produced to greater yields and administered more reproducibly than the known heterogeneous clusters. The crystal compositions of the present invention are pharmaceutically attractive because they are relatively uniform and remain in suspension for a longer period of time than the crystalline clusters or s amorphous crystalline suspensions which tend to settle rapidly, aggregate or clump together, clog syringe needles and generally exacerbate unpredictable dosing. Most importantly, the crystal compositions of the present invention display extended, uniform, and reproducible pharmacokinetics which can be modulated by adding zinc using conventional crystal soaking techniques or, alternatively, by including zinc in the crystallization solution.